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Biomedical subjects

H E Broxmeyer

Publications and source records attributed to H E Broxmeyer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-alpha and transforming growth factor-beta1.

The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.

Antigens, CD↗
PubMed1999

TECK, an efficacious chemoattractant for human thymocytes, uses GPR-9-6/CCR9 as a specific receptor.

Chemokines regulate leukocytes trafficking in normal and inflammation conditions. Thymus-seeding progenitors are made in bone marrow and migrate to the thymus where they undergo their maturation to antigen-specific T cells. Immature T cells are in thymic cortex, while mature thymocytes are in medulla. Chemokines may be important for homing of thymus-seeding progenitors, and/or differential thymocyte localization in thymus. Here we report that GPR-9-6, now called CC chemokine receptor 9 (CCR9), is a receptor for thymus-expressed chemokine, TECK. Among a panel of chemokines tested, TECK specifically induced calcium flux in CCR9-expressing cell lines. We also showed that TECK efficaciously induced chemotaxis of immature CD4(+)CD8(+) double-positive, and mature CD4(+) and CD8(+) single-positive human thymocytes. Our data suggest that TECK/CCR9 interaction may play a pivotal role in T-cell migration in the thymus.

Amino Acid Sequence↗
PubMed1999

Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.

Actins↗
PubMed1999

Flt3 ligand antitumor activity in a murine breast cancer model: a comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action.

We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing Flt3L (C3Lt-Flt3L) and GM-CSF (C3L5-GMCSF) by injecting 1 x 10(4) cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 x 10(3) cells; or low, 1 x 10(3) cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5-Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.

Animals↗
PubMed1999

Dominant myelopoietic effector functions mediated by chemokine receptor CCR1.

Macrophage inflammatory protein (MIP)-1alpha, a CC chemokine, enhances proliferation of mature subsets of myeloid progenitor cells (MPCs), suppresses proliferation of immature MPCs, and mobilizes mature and immature MPCs to the blood. MIP-1alpha binds at least three chemokine receptors. To determine if CCR1 was dominantly mediating the above activities of MIP-1alpha, CCR1-deficient (-/-) mice, produced by targeted gene disruption, were used. MIP-1alpha enhanced colony formation of marrow granulocyte/macrophage colony-forming units (CFU-GM), responsive to stimulation by granulocyte/macrophage colony-stimulating factor (GM-CSF), and CFU-M, responsive to stimulation by M-CSF, from littermate control CCR1(+/+) but not CCR1(-/-) mice. Moreover, MIP-1alpha did not mobilize MPCs to the blood or synergize with G-CSF in this effect in CCR1(-/-) mice. However, CCR1(-/-) mice were increased in sensitivity to MPC mobilizing effects of G-CSF. Multi-growth factor-stimulated MPCs in CCR1(-/-) and CCR1(+/+) marrow were equally sensitive to inhibition by MIP-1alpha. These results implicate CCR1 as a dominant receptor for MIP-1alpha enhancement of proliferation of lineage-committed MPCs and for mobilization of MPCs to the blood. CCR1 is not a dominant receptor for MIP-1alpha suppression of MPC proliferation, but it does negatively impact G-CSF-induced MPC mobilization.

Animals↗
PubMed1999

Isolation of ALP, a novel divergent murine CC chemokine with a unique carboxy terminal extension.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report here, the isolation of a novel murine CC chemokine termed ALP for its amino terminal peptide sequence. This novel chemokine is distantly related to other CC chemokines (37% identity with murine Exodus-1/LARC/Mip-3alpha), but has a unique carboxy terminal extension. It is expressed preferentially in testis, heart, and liver, which is atypical for CC chemokines.

Amino Acid Sequence↗
PubMed1999

Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma.

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.

Antibodies, Monoclonal↗
PubMed1999

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors.

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.

CD56 Antigen↗
PubMed1999

Amphotropic retrovirus transduction of hematopoietic stem cells.

Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.

Adult↗
PubMed1999

Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors.

Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.

Animals↗
PubMed1999

Enhanced myeloid progenitor cell cycling and apoptosis in mice lacking the chemokine receptor, CCR2.

Chemokines regulate hematopoiesis in part by influencing the proliferative status of myeloid progenitor cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin- populations of BM from CCR2-deficient (-/-) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with progenitors from spleens of CCR2 (-/-) mice. Increased cycling status of CCR2 (-/-) BM MPC did not result in increased numbers of nucleated cells or MPC in BM or spleens of CCR2 (-/-) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin- BM cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin- population of BM cells from CCR2 (-/-) mice had a significantly higher percentage of apoptotic cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin- cells. The increased percentage of apoptotic c-kit+lin- cells was due to elevated apoptosis within the c-kitdimlin-, but not the c-kitbrightlin-, subpopulations of cells. Consistent with enhanced apoptosis of phenotypically defined cells, MPC from CCR2 (-/-) BM and purified c-kit+lin- cells demonstrated decreased cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of progenitor cells in the BM, but also enhance survival of these cells.

Animals↗
PubMed1999

H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.

The proto-oncogene product, p21(ras), has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; alpha4beta1 integrins) and VLA-5 (alpha5beta1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfected Baf3 cells. Anti-beta1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other types of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti-phospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.

Animals↗
PubMed1999

Cloning of BRAK, a novel divergent CXC chemokine preferentially expressed in normal versus malignant cells.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.

Amino Acid Sequence↗
PubMed1999

Thrombopoietin-induced conformational change in p53 lies downstream of the p44/p42 mitogen activated protein kinase cascade in the human growth factor-dependent cell line M07e.

Thrombopoietin is a cytokine with potent megakaryocytopoietic and thrombopoietic activities in vivo. Wild-type p53 is a conformationally flexible, anti-oncogenic transcription factor that plays a principal role in mediating growth factor withdrawal-induced apoptosis in factor-dependent hematopoietic cells. We recently reported that Tpo induces a conformational change in and functional inactivation of p53, coincident with its anti-apoptotic effects, in the human factor-dependent cell line M07e. In an effort to identify potential signaling cascades through which Tpo illicits these effects on p53, we report here that treating M07e cells with MAPK kinase inhibitor PD98059 dramatically suppressed Tpo-induced conformational change in p53 as well as Tpo-enhanced viability in M07e cells in a p53-dependent manner. Furthermore, the expression of constitutively active Raf1 in M07e cells induced conformational change in p53 independent of Tpo stimulation. Inhibition of the JAK/STAT pathway revealed that JAK/STAT signaling plays an insignificant role in conformational modulation of p53 and apoptosis suppression. Inhibition of phosphatidylinositol-3 kinase did not have a significant effect on p53 conformation but did have a weak but significant effect on Tpo-enhanced viability. Cytokine-induced activation of the MAPK pathway and the subsequent functional neutralization of p53, may be an event by which apoptosis is commonly suppressed in hematopoiesis.

Animals↗
PubMed1999

p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells.

A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21(cip-1/waf-1). Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21(cip-1/waf-1) deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21(cip-1/waf-1) expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21(cip-1/waf-1) in p21(cip-1/waf-1) antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.

Cell Cycle↗
PubMed1999

p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells.

Flt3/Flk2 belongs to class III receptor tyrosine kinases. Like other members of type III receptor tyrosine kinases, murine Flt3 induces tyrosine phosphorylation of p85 and subsequently activation of PI3 kinase upon FL binding. While p85 binds murine Flt3 at Y958 in the carboxyl terminus of the receptor, human Flt3 does not have a potential p85-binding site in the carboxyl terminus. In this study, we examined whether p85 binds to human Flt3 in Baf3/Flt3 and THP-1 cells. In contrast to murine Flt3, p85 is not tyrosine phosphorylated after FL stimulation, nor does it bind Flt3 in both cell lines. Instead p85 associates inducibly with tyrosine phosphorylated SHP-2 and constitutively with SHIP and two tyrosine phosphorylated proteins with molecular weights about 100-kDa (p100) and 120-kDa (p120) in Baf3/Flt3 cells. The p100 associates with both p85 and SHP-2. In THP-1 cells, p85 associates inducibly with tyrosine phosphorylated SHIP, p100 and p120. These results indicate that p85 does not bind human Flt3, but forms a complex with SHP-2, SHIP, p100 and p120 in hematopoietic cells.

Amino Acid Sequence↗